The Sxr (Sex reversed) region is a small fragment of the mouse Y chromosome short arm, transposed distal to the pairing/recombination region of the Y in XYSxr mutant mice. During male meiosis, Sxr is regularly transferred to the paternal X chromosome, giving rise to sterile XXSxr males. It has been shown that this region contains the genes controlling primary sex determination, Tdy; H-Y antigen expression, Hya (as defined by T cell killing and proliferation assays); a spermatogenesis gene, Spy; the Zinc finger genes Zfy-1 and Zfy-2 and Sry a prime candidate for Tdy. In addition we have compelling evidence for a new conserved Sxr gene with both X and Y linked copies, detected by an Sxr probe pY8. A deletion mutant of Sxr termed Sxrb has been described which has lost Zfy-2 sequences and Hya and Spy functions. Hence, both Hya and Spy are contained within the deletion and are now amenable to molecular analysis and cloning. The thrust of this (revised) grant proposal is (1) to construct a long-range map the Sxr region; (2) to produce a high resolution contig map of the entire Sxrb deletion using overlapping YAC clones; (3) to clone Hya and investigate its relationship to Spy using transgenic technology, and (4) to characterize the new Y-X common gene. YACs spanning the Sxrb deletion will be transfected into an XXSxrb (H-Y negative) cell line and colonies screened with cloned proliferative helper and killer lines for the expression of H-Y. Positive clones containing the Hya gene will be analyzed for their DNA content with Sequence Tagged Site (STS) probes taken from the relevant YAC clone and exonic sequences of Hya sought using species conservation, etc. The cloning of Hya will allow us to investigate its specific function and more broadly the role of the some 30 minor transplantation antigens that have been identified in both mouse and man. A new fundamental field of study could thus be opened up. The cloning of the new Sxr gene(s) from the Y and X, initially detected with Sxr probe pY8 and a detailed analysis of this finding, is new and relevant to the field as a whole. The program itself is designed to generate as much valuable scientific information as possible. The isolation and characterization of overlapping YAC clones covering the entire Sxrb deletion and the isolation of evenly spaced STS's will give a high resolution map of this important region and enable us to examine the strong possibility that there may exist as yet unknown genes in this area of the Y. The long-range map of the Sxr region is essentially a map of the short arm of the mouse Y, containing a minimum of six important genes. In addition to providing essential data on the size of the Sxrb deletion, all chromosomal maps such as these will be of central importance in the wider context of the "genome project".